The goal of the proposed research is to understand the biochemical processes which control differentiation of erythroid cells. The method of approach will be to analyze various properties of a permanent Friend Leukemia Virus-transformed erythroleukemic cell line which exhibits a characteristic erythroid differentiation when cultured in vitro under appropriate conditions. By comparing the control of differentiation of these malignant cells with available data on the control of normal differentiation we hope to understand if transformation with leukemic viruses alters these control processes. We will determine what controls are exerted in these cells during differentiation on the transcription, processing and translation of globin messenger RNA. The properties of globin mRNA sequences present in the nucleus and cytoplasm will be examined. Globin mRNA sequences will be assayed by DNA-RNA hybridization with a DNA copy of globin mRNA synthesized with RNA dependent DNA polymerase. Studies with whole cells will be complemented by experiments designed to study the transcription and processing of globin mRNA sequences in cell-free extracts. We will attempt to purify the globin gene sequences from cellular DNA. The other major area of investigation involves the use of cellular genetics for analyzing the regulatory processes controlling erythroid differentiation. The properties of cell hybrids (some of which we have already prepared) formed between erythroleukemic cells and other mouse and human cell lines will be examined in order to: a) determine the biochemical basis of extinction of differentiated functions in somatic cell hybrids, and b) assign the human loci specifying globin chains to particular human chromosomes. A second approach will be to isolate mutant erythroleukemic cells which are unable to differentiate. The mutants will be analyzed biochemically for the presence of several phenotypic markers of erythroid differentiation and studied further by complementation analysis using cell hybrids formed between mutants and parental cells and amongst mutants.